Suppression of bone resorption by quinolines

ABSTRACT

The present invention focuses upon a method for inhibiting bone resorption. This method involves administering a 5-lipoxygenase inhibitor to a subject in an amount inhibiting the effects of an osteoclast-stimulating factor. When the production of the osteoclast-stimulating factors such as PTH, PTHrp, IL-1, TNF, LT, 1,25(OH) 2  D 3  or other factors which may stimulate the production of 5-LO metabolites via the 5-lipoxygenase pathway is inhibited, bone resorption markedly declines. The direct osteoclast-stimulating factors include leukotriene, peptidoleukotriene and 5-hydroxyeicosatetraenoic acid. Other factors yet to be identified or previously known may also be 5-lipoxygenase metabolites that stimulate bone resorption. While 5-lipoxygenase inhibitors may be substrate analogs or allosteric inhibitors, a substance which inhibits the activity of this enzyme may utilize other mechanisms (e.g., inhibition of 5-LO biosynthesis) and nevertheless function to inhibit bone resorption. Preferred inhibitors included NGDA, MK886 and ZM230,487. The best inhibitor thus far noted is ZM230,487. The inhibition of bone resorption is highly desirable with, for example, periodontal disease, osteoporosis, estrogen deficiency, Paget&#39;s disease, inflammatory bone loss, bone malignancy, hyperparathyroidism. The preferred range of 5-lipoxygenase inhibitors administered is from 0.1 to 10 mg/kg body weight/day.

The United States government has rights in the present invention becauserelevant research was supported by Grant NIH-POI AR 39529.

BACKGROUND OF THE INVENTION

Diseases associated with bone loss are usually accompanied by increasedosteoclast activation. Such diseases include estrogen deficiency afterthe menopause, osteoporosis, primary hyperparathyroidism, malignancy,Paget's disease of bone and periodontal disease. The bone loss is causedby osteoclast activity. Osteoclasts are unique multinucleated cellswithin bone that are responsible for bone degradation. These are theonly cells in the body known to be capable of resorbing bone. Sincediseases of bone loss are associated with increased activity of thesecells, it is important to understand the mechanisms by which osteoclastsare activated in these disease states, and to devise rational andtherapeutic means to inhibit this activation.

The molecular mechanisms by which osteoclasts are activated are unknown.In vitro data indicate cytokines and systemic hormones with boneresorbing effects do not act directly on osteoclasts, but rather act onaccessory cells in the bone marrow microenvironment and that these cellsin turn are responsible for osteoclast activation (Rodan & Martin 1981,McSheehy & Chambers 1986). This activation may be mediated either bycell-cell contact or by locally active soluble factors. In a search forcell sources of such soluble factors, the present inventors found that astromal cell line (C433) derived from a giant cell tumor of boneproduced prodigious amounts of osteoclast-stimulating activity greaterthan any we found in conditioned media from cells with osteoblastcharacteristics (Oreffo et al., 1993).

Human giant cell tumors of bone comprise heterogeneous cell populations,including giant cells with many of the phenotypic and functionalcharacteristics of osteoclasts as well as mononuclear cells. Themultinucleated cells are positive for osteoclast surface antigens(Davies et al, 1989), for tartrate-resistant acid phosphatase (TRAP),¹possess receptors for calcitonin (Komiya et al., 1990) and lackmonocyte-macrophage surface antigens (Goldring et al., 1986). Themononuclear cells comprise two distinct populations. One population doesnot persist in culture and is positive for Ia and monocyte-macrophageantigens (Ling et al., 1988). Another population persists in culture andresembles connective tissue stromal cells, produces Types I and IIIcollagen, and has receptors for parathyroid hormone (Goldring et al.,1986). These latter cells can be readily established in cell culture.One cell line (C433) derived from stomal cells from a giant cell tumoris shown herein to cause greater increases in osteoclast activity asmeasured by accumulation of TRAP activity than any of the knownosteoblast-like cell lines. This study concerns characterizing theosteoclast stimulating activity produced by this cell line. Thisactivity is ascribed to 5-hydroxyeicosanoids, which are 5-lipoxygenasemetabolites of arachidonic acid.

Dziak and co-workers (Mohammed et al., 1989) examined the role ofleukotrienes in orthodontic tooth movement, a model used to examine boneremodeling. These investigators found significant inhibition of toothmovement using the leukotriene inhibitor AA 861, even though enhancedlevels of prostaglandins were detected in this treated tissue. Theysuggested that inhibition of LT synthesis might influence tooth movementand that prostaglandins and leukotrienes might mediate different stepsin a cascade of events that results in initiation of bone remodeling.

5-lipoxygenase metabolites possess a diverse range of biologicalactivities, especially in allergic and inflammatory responses. Themolecule LTB-4 is chemotactic for polymorphonuclear leukocytes,eosinophils, lymphocytes, and monocytes and will increase adherence,oxygen radical production, and lysosomal degranulation inpolymorphonuclear leukocytes (Goldman et al., 1986). LTC-4 and LTD-4have been shown to promote myeloid colony formation (Ziboh et al.,1986), proliferation of glomerular epithelial cells (Baud et al., 1985),and secretion of luteinizing hormone and luteinizing hormonereleasing-hormone (Parker 1987). A number of cytokines and growthfactors such as interleukin-1 and interleukin-2 will induce productionand secretion of leukotrienes (Parker 1987). Significant breakthroughshave been made in identifying proteins and enzymes involved in thesynthesis of these compounds. For example, only cells which contain both5-lipoxygenase (5-LO) and a recently cloned protein,5-lipoxygenase-activating protein (FLAP), will produce leukotrienes(Dixon et al., 1990; Reid et al., 1990). Hormones which induce 5-LOmetabolite production may regulate not only 5-LO enzymes but FLAPexpression.

There are previous reports that leukotrienes (LT) may be modulators ofbone cell function. Meghji et al (1988) tested purified leukotrienes inthe neonatal mouse calvarial assay and found significant boneresorption. However, other investigators have not been able to repeatthese results (personal communications). This may be due to the unstablenature of these compounds. The present inventors now report that specialprecautions are necessary to maintain biological activity. The compoundsmust be stored under argon in the absence of light. Once removed fromthese conditions the commercial compounds must be diluted quickly andused immediately. Activity of commercially available compounds wasdetectable at higher concentrations (10⁻⁶ to 10⁻⁷ M) in the organculture assay, whereas activity was detectable at much lowerconcentrations (10⁻¹⁰ to 10⁻¹¹ M) in the isolated avian osteoclast andisolated human giant cell assays. This may reflect readier access of5-LO metabolites to target cells in the isolated cell culture system.

Thus, the mechanisms by which leukotrienes and other systemic factorsand hormones are responsible for osteoclast activation remain unknown.The connection between leukotrienes and other systemic hormones and boneresorbing factors and whether their effects on osteoclasts are relatedor independent was also unknown prior to the information described bythe present application.

Inhibitors of 5-Lipoxygenase used herein include:

NGDA--nordihydroguaiaretic acid (see structure in FIG. 9);

MK886--(see structure in FIG. 10); and

ZM230,487--(see structure in FIG. 11)

SUMMARY OF THE INVENTION

The present invention focuses upon a method for inhibiting boneresorption. This method involves administering a 5-lipoxygenaseinhibitor to a subject in an amount suppressing production of anosteoclast-stimulating factor. When the production of theosteoclast-stimulating factors via the 5-lipoxygenase pathway isinhibited, bone resorption markedly declines. The osteoclast-stimulatingfactors include peptido-leukotriene and 5-hydroxyeicosatetraenoic acid.Other factors yet to be identified or previously known may also be5-lipoxygenase metabolites that stimulate bone resorption. While5-lipoxygenase inhibitors may be substrate analogs or allostericinhibitors, a substance which inhibits the activity of this enzyme mayutilize other mechanisms (e.g., inhibition of 5-LO biosynthesis) andnevertheless function to inhibit bone resorption. Preferred inhibitorsincluded NGDA, MK886 and ZM230,487. The best inhibitor thus far noted isZM230,487.

The inhibition of bone resorption is highly desirable with, for example,periodontal disease, osteoporosis, estrogen deficiency, Paget's disease,inflammatory bone loss, bone malignancy, hyperparathyroidism.Administering of a 5-lipoxygenase inhibitor may be enteral--when oraladministration is desired, parenteral, when appropriate (preferably byvascular injection or infusion), or topical such as application to oraltissues to prevent bone loss due to periodontal disease. The preferredrange of 5-lipoxygenase inhibitors administered is from 0.1 to 10 mg/kgbody weight/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Quantitation of pit formation on sperm whale dentine by ratosteoclasts treated with C433-conditioned medium (DMEM, 0.1% BSA, 10⁻⁸ M1,25-(OH)₂ vitamin D₃), with parathyroid hormone (PTH) (10⁻⁸ M) andC=control (DMEM, 0.1% BSA, 10⁻⁸ M 1,25-(OH)₂ vitamin D₃) (FIG. 1A) orhuman giant cells treated with C433-conditioned medium (FIG. 1B).Quantitation of TRAP activity by avian osteoclasts incubated withC433-conditioned medium (FIG. 1C) and human giant cells incubated withC433-conditioned medium (FIG. 1D). T/C=treated/control ratio.

FIG. 2. Molecular mass determination of C433 activity. Fifty ml ofC433-conditioned medium was filtered, lyophilized, and reconstitutedbefore applying to a Bio-Gel P-2 column equilibrated in 10 mM ammoniumbicarbonate, pH 7.1, and standardized (insulin >2000 daltons;nonapeptide, 986 daltons; and sodium chloride, 58.5 daltons). Proteinwas monitored at 280 nm. Every second fraction was bioassayed utilizingthe isolated avian osteoclast TRAP assay. Molecular mass of the activitywas determined to be less than 1000 daltons. MUP, methylumbelliferylphosphate.

FIGS. 3A-3D. Chemical characterization of activity. FIG. 3A.C433-conditioned medium was treated with ultraviolet light (UV) for 24 hor placed in a boiling water bath (100° C.) for 15 min and bioassayed.FIG. 3B. amino peptidase M (A), carboxypeptidase (C), or Pronase-CB (P)were incubated in the presence of C433-conditioned medium for 12 h at37° C. FIG. 3C. 433 cells treated with flurbiprofen (FLUR), 10⁻⁶ Mindomethacin (INDO) 10⁻⁶ M, or 10⁻⁵ M nordihydroguaiaretic acid (NDGA).The resultant conditioned medium was bioassayed for activity. FIG. 3D.C433 were treated with 10⁻⁶, 10⁻⁵ or 10⁻⁴ M nordihydroguaiaretic acid.Conditioned medium was bioassayed for induction of TRAP activity inavian osteoclasts.

FIGS. 4A-4C. Partial purification of biological activity fromC433-conditioned medium using C₈ reverse phase high pressure liquidchromatography (FIG. 4A). The gradient was 0-100% methanol, 1% aceticacid over 60 min with 6-ml fractions collected. The fractions were driedunder nitrogen, and those fractions to be used for bioassay (TRAPinduction in avian osteoclasts) were stabilized with 20 μl of 10% bovineserum albumin. Fractions to be used for GC-MS analysis were immediatelyderivatized. FIG. 4A. Commercially available (Sigma) LTC₄ /LTD₄ (1.2 μg)and 5-HETE (5 μg) were applied to a C₈ reverse phase HPLC under theexact conditions as C433-conditioned medium. FIG. 4B. LTC₄ /LTD₄standard eluted at the same fraction as activity 2 whereas FIG. 4C5-HETE standard (85% methanol) eluted as activity 3. MUP,methylumbelliferyl phosphate.

FIG. 5. Stable isotope dilution/gas chromatography-mass spectrometryanalyses of HPLC fractions exhibiting osteoclast activating activity.[¹³ C₄ ]LTC₄ (0.5 ng) was added to each fraction which was thenhydrogenated and converted to the pentafluorobenzyl ester trimethylsilylether derivative. Ions monitored during GC-MS analysis corresponded tothe product of the endogenous 5-LO metabolites (m/z 399) and theinternal standard (m/z 403). The slightly shorter retention timeobserved during analysis of the activity 1 fraction reflected a minordifference in analytical conditions. The relative abundance of the m/z399 ion was normalized for each analysis to the relative abundance ofm/z 403. For quantification, response rations (m/z 399 to m/z 403) werebased on peak area calculation.

FIGS. 6A-6B. UV-treated C433-conditioned medium (CM) stabilized theHPLC-purified activity 2 and activity 3 fractions as determined in theisolated rat osteoclast pit formation assay.

FIGS. 7A-7D. Biological effects of commercially availablepeptido-leukotrienes and 5-HETE. LTC₄ /Ltd₄ (10⁻¹⁰ M) and 5-HETE (10⁻¹⁰M) were incubated for 48 h in the presence of isolated avian osteoclasts(FIG. 7A), isolated 23C6(+) human giant cells of bone (FIG. 7B), andTRAP induction was measured. LTC₄ (10⁻¹⁰ M) was tested on pit formationby isolated 23C6+ human giant cells (FIG. 7C) and LTE₄ (10⁻¹⁰ M) on pitformation by isolated rat osteoclasts (FIG. 7D). *, significantdifference using Bonferonni (p<0.05). PTH, parathyroid hormone.

FIG. 8. Effect of combining commercially available 5-HETE and thepeptido-leukotriene LTD₄ on TRAP activity in isolated avian osteoclasts.Both 5-HETE and LTD₄ optimally stimulated TRAP activity at 10⁻¹⁰ M.Higher molarity (10⁻⁸ M) appears to stimulate suboptimally. The effectsof combining 5-HETE and LTD₄ appear additive.

FIG. 8A shows the effect of LTB₄ on ⁴⁵ Ca release in the mouse calvarialassay.

FIG. 9 shows the structure of NDGA.

FIG. 10 shows the structure of MK886.

FIG. 11 shows the structure of ZM 230,487(1-ethyl-6-[fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl)phenoxy]methyl-quinol-2-one).

FIG. 12A shows the effects of NDGA (10⁻⁶ M), MK886 (1 μg/ml) and ZM230,487 (10 ng/ml) on isolated rat osteoclasts. The cells were culturedin the presence of factor and 5-LO inhibitor for 20 hours beforeharvest. Data are expressed in number of pits per dentine slice.

FIG. 12B shows data expressed as area of resorption lacunae per dentineslice.

FIG. 12C shows the number of resorption lacunae per osteoclasts.

FIG. 12D shows the area of resorption lacunae per osteoclast.

FIGS. 13A-13B show that ZM 230,487 significantly inhibits resorption dueto 1,25(OH)₂ vitamin D₃. Significance was not reached in this experimentwith WR14 LPS. FIG. 13(A) shows the number of resorption lacunae perosteoclast. FIG. 13(B) shows the area of resorption lacunae perosteoclast.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises (1), a method for treating osteoporosisby inhibition of osteoclastic bone resorption, (2), a method fortreating Paget's disease of bone by inhibition of osteoclastic boneresorption, (3), a method for treating metastatic cancer in bone byinhibition of bone resorption, (4), a method of treating periodontaldisease by inhibition of bone resorption, and (5), a method for treatingother diseases associated with increased osteoclastic bone resorption.Bone resorption may be inhibited by inhibitors to show the production ofmetabolites stimulating osteoclasts. While a number of 5LO inhibitorsare described herein, it is understood that many others are possible.The assays for such inhibitors described herein enable others to bereadily developed.

For each of these diseases, 5-lipoxygenase inhibitors may be enterallyor parenterally administered. The doses, duration of treatment andtiming of administration would need to be determined by Phase I clinicalstudies, but based on in vitro data should be in the range of 0.1-10mg/kg/day. The compound could also be applied topically such as creamsor toothpaste for periodontal disease.

Bone resorption requires cooperation between osteoclasts and mononuclearaccessory cells by mechanisms which have not been elucidated.Multinucleated cells in giant cell tumors of bone have many phenotypicand functional characteristics of normal osteoclasts. The interactionbetween the bone-resorbing multinucleated cells and the distinctmononuclear stromal cells from these tumors was examined. Thesemononuclear cells produce an activity which stimulates both giant cellsfrom giant cell tumors and rodent osteoclasts to resorb bone in vitro.The activity has been identified and found that it represents severalproducts of the 5-lipoxygenases pathway of arachidonic acid metabolism,for example, 5-hydroxyeicosatetraenoic acid and the leukotrienes. Asdescribed herein, 5-lipoxygenase metabolites stimulate isolatedosteoclasts to resorb bone in vitro and represent a mechanism by whichmononuclear stromal cells in human giant cell tumors communicate withthe giant cells. In addition, the results desorbed herein explain onemechanism for communication between accessory cells and osteoclastsinvolved in normal bone resorption.

The present inventors show that leukotriene compounds, in addition toother systemic hormones such as parathyroid hormone and 1,25dihydroxyvitamin D₃ and cytokines such as interleukin-1 and tumornecrosis factor, are capable of inducing bone resorption both in vitroand in vivo (Gallwitz et al, 1993). This was shown by studies on thecells isolated from human giant cell tumors of bone. The giant cells andgiant cell tumors of bone resemble osteoclasts, but other cells in thesetumors produce factors which activate the osteoclasts. These factorshave been identified and it was found that they represent severalproducts of the 5 lipoxygenase pathway of arachidonic acid metabolism,the leukotrienes. The data indicate that these 5 lipoxygenasemetabolites stimulate isolated osteoclasts to resorb bone in vitro.

The following examples illustrate the best mode and technical backgroundof the present invention. They should not limit the claims of thepresent invention unless otherwise specified.

EXAMPLE 1 5-Lipoxygenase Metabolites of Arachidonic Acid StimulateIsolated Osteoclasts to Resorb Calcified Matrices EXPERIMENTALPROCEDURES

Materials

The radioimmunoassays for 5-HETE were purchased from Advanced Magnetics,Inc. (Cambridge, Mass.). The commercially available 5-HETE andleukotrienes, protease enzymes, flurbiprofen, indomethacin, andnordihydroguaiaretic acid were purchased from Sigma.

Isolation of Avian Osteoclasts

Avian osteoclasts were isolated from medullary bone of laying WhiteLeghorn hens Gallus domesticus (Pioneer Animal Supply, Kingswheel, Ohio)as described previously by Zambonin-Zallone and Teti (1981). In brief,bone marrow suspensions from the medullary bone of femora were filteredthrough Nytex cloth (110 μm, t'etko, Elmsford, N.Y.), centrifuged for 5min at 1200 rpm, and the cell pellet resuspended in 0.2% NaCl for 3 minto lyse erythrocytes. After layering the cells on 100% fetal bovineserum for 1 h, sedimented cells were further filtered through Nytexfilters (55 μm). Cells were harvested, resuspended in α-minimal Eagle'smedium (GIBCO) containing 10 fetal bovine serum, with penicillin (100units/ml), streptomycin (100 μg/ml), and Ara-C, to inhibit proliferationof nonosteoclastic cells. Cells were plated in 24- or 48-well plates(Costar, Cambridge, Mass.) at 1×10⁴ cells/well and incubated at 37° C.in 10% CO₂ humidified air for 48 h, after which they were washed toremove nonadherent cells. When matured osteoclasts were observedexperiments were begun and terminated within 48 h.

Quantitation of Tartrate-resistant Acid Phosphatase by FluorescenceSpectroscopy

Osteoclast TRAP activity was measured using fluorescence spectroscopy asdescribed by Chambers et al. (1987) with minor modifications. In brief,media from osteoclast cultures were harvested and stored at -70° C.until ready for assay.

The cells were washed with phosphate-buffered saline and harvested in0.5 ml of Triton X-100 (0.05%, w/v). Aliquots of media or lysate 30 μl)were incubated with 170 μl of 2 mM methylumbelliferyl phosphate, pH 5.0,in 0.48M acetate buffer (0.48M sodium acetate, 0.48M acetic acid, pH5.0) and 30 mM tartaric acid. Samples were incubated for 30 min at 37°C. and the reaction terminated with 100 μl of stop solution containing50 mM glycine, 50 mM EDTA, pH 10.4. Fluorescence was measured atexcitation 360 nm and emission 448 nm using a fluorimeter (Fluoroskan,Flow Instruments). Enzyme activity was expressed as micromoles ofmethylumbelliferyl phosphate hydrolyzed/min/μg of protein and theprotein content measured by the technique of Lowry et al. (1951).

Disaggregated Neonatal Rat Osteoclast Pit Formation Assay

Quantitation of the effects of isolated osteoclasts on calcifiedmatrices was determined using minor modifications of the disaggregatedosteoclast resorption assay as described by Boyde et al. (1984). Spermwhale dentine (0.25×7×7 mm) was prepared using a Buehler low speeddiamond saw (Buehler, Lake Bluff, Ill.) followed by sonication (15 min)in several changes of distilled water. Slices were sterilized usingultraviolet light.

Neonatal Sprague-Dawley rats (2-3 days) were sacrificed by decapitation,the femurs and tibias were removed, scraped free of adherent tissue, andtrimmed free of the epiphyses. The bones from one litter were combinedand quickly minced using a scalpel blade in 2 ml of 199 medium and thenvigorously mixed with a pipette in an 8-ml tube, allowed to settle for10 s, and then 100 μl of the suspension was added to each well for atotal of 16 wells in a 48-well microtiter plate containing sperm whaledentine (approximately four neonates are used per 16 wells). The cellswere incubated at 37° C. for 30 min at which time the dentine wasremoved, washed in media, and placed in a fresh 48-well plate containing250 μl of test medium.

Osteoclasts were stained for TRAP and counted and then pit numbers werecounted following toluidine blue (0.1% w/v) staining by lightmicroscopy. The plan area of matrix resorbed was quantitated using acomputer-assisted morphometric program on a Bioquant System IV analysissystem (R & M Biometrics, Nashville, Tenn.).

Preparation of 23C6 Positive Cells from Giant Cell Tumors

Human giant cell tumors (normally discarded after surgery) were mincedand passed through 1#40SS wire mesh screen, allowed to settle for 5 min,and the cell suspension above the sediment collected. The cells wereincubated with 23C6 monoclonal antibody that identifies the osteoclastvitronectin receptor (generously provided by Dr. Michael Horton) (1:10dilution of hybridoma supernatant/10⁶ cells) at 4° C. in serum-freemedium for 30 min, after which immunomagnetic beads coated withanti-mouse IgG (Dynabead, Dynal, Inc., Great Neck, N.Y.) were added tothe cell suspension. The cell suspension was mixed for 5 min and thenthe 23C6 positive cells were separated using a magnet (Dynal, Inc.) onthe side of the tube while suction was applied to remove all negativecells. Over 90% of the cells adhering to the magnetic beads were23C6-positive, and these cells were used for induction of TRAP activityand for pit formation on sperm whale dentine. Cells were plated at 60cells/dentine slice or 4×10⁴ cells/well in 24-well plates for inductionof TRAP activity.

Neonatal Mouse Calvarial Assay

The assay was performed as described by Gowen et al. (1983). Timedpregnant mice were injected with ⁴⁵ Ca 2 days before parturition.Half-calvaria were removed from the 1-2-day-old pups and preincubatedfor 24 h in BGJb medium at 37° C. in a humidified atmosphere of 5% CO₂before transfer to fresh media with or without test substances for 48 h.The bones were incubated for a further 72 h and media and bonescollected. Bone-resorbing activity was expressed as the percentage ofthe total ⁴⁵ Ca released into the medium.

Purification Protocol for the C433 Active Fractions

C433 cells were grown in 10% fetal bovine serum (Whittaker,Walkersville, Md.) 50% RPMI, 50% McCoys (Flow, McLean, Va.) until adensity of 10⁴ cells/ml was obtained, at which time the cells wereharvested, washed, and placed in serum-free DMEM plus 10⁻⁸ M, 1,25-(OH)₂D₃ for 48 h before harvest of conditioned medium. The conditioned mediumwas brought up to 15% ethanol and acidified to pH 3.5 with concentratedHCl. This conditioned medium was applied to a prewetted C₁₈ Sep-Pak(Millipore, Waters, Milford, Mass.), and bound material was extractedusing ethyl acetate. This material was dried under nitrogen andresuspended in water, 1% acetic acid and analyzed by high pressuremeasure liquid chromatography (HPLC) (Waters, Milford, Mass.) applied toa C₈ semipreparative 25 cm×10 mm reverse phase column (KeystoneScientific, Inc., Bellefonte, Pa.) at 2 ml/min. The gradient was 0-100%methanol, 1% acetic acid over 60 min with 6-ml fractions collected. Thefractions were dried under nitrogen, and those fractions to be assayedfor osteoclast stimulating activity were stabilized with 20 μl of 10%bovine serum albumin. Fractions to be used for gas chromatography-massspectrometric analysis were prepared for derivatization.

Gas Chromatography-mass Spectrometric Analysis for 5-LipoxygenaseProducts

Bioactive HPLC fractions were analyzed for the presence of5-lipoxygenase products using a modification of the procedure describedby Balazy and Murphy (1986). Each fraction was supplemented with[8,9,20,11-¹³ C₄ ]LTC₄ (Raftery et al., 1992) and hydrogenated using arhodium black catalyst. This procedure yields a common product,5-hydroxyeicosanoic acid, from 5-lipoxygenase products such as 5-HETEand the peptido-leukotrienes. Each hydrogenated sample was converted tothe pentafluorbenzyl ester trimethylsilyl ether derivative. Gaschromatographic-mass spectrometric analyses were performed with selectedion monitoring of ions characteristic of derivative of5-hydroxyeicosanoic acid and of the [¹³ C₄ ] analogue.

Statistical Analysis

Data were analyzed using the Student's t test or the Bonferonni test(p<0.05) using a statistical package for the IBM PC, SAS Industries,Inc. (Cang, N.C.).

RESULTS

The conditioned media harvested from C433 cells stimulated freshlyisolated neonatal rat osteoclasts to form resorption lacunae on spermwhale dentine. There was a 3-fold increase in resorbed area/dentineslice compared with controls, and the C433-conditioned medium containedsimilar resorbing activity to maximal concentrations of parathyroidhormone (10⁻⁸ M) in this assay (FIG. 1A.). The conditioned medium alsostimulated giant cells isolated from human giant cell tumors of bone toform resorption lacunae on sperm whale dentine. These giant cells wereisolated using a panning technique with an antibody, 23C6, whichrecognizes osteoclasts preferentially (Horton et al., 1985). The area ofdentine resorbed by these giant cells was increased 180-fold overcontrols (FIG. 1B.). When conditioned medium from C433 cells wasincubated with organ cultures of neonatal mouse calvariae previouslyincorporated with ⁴⁵ Ca, there was an increase in bone resorption. Thus,the conditioned medium harvested from C433 cells contains an activitywhich stimulates isolated osteoclasts to form resorption lacunae andstimulates bone resorption in organ cultures.

In order to characterize and identify the bone-resorbing activityproduced by C433 cells, Tartrate-Resultant Acid Phosphatase (TRAP)activity in isolated purified avian osteoclasts was used as a measure ofosteoclast stimulation. Measurement of pits on calcified matrices istime-consuming, difficult and imprecise, and is not suitable for apurification assay. TRAP activity has been used previously as aparameter of osteoclast stimulation (Chambers and Fuller, 1984; Zaidi etal., 1988; Oreffo et al., 1990, 1992). This measurement was used toassess the capacity of C433-conditioned medium to activate osteoclasts.Increases in TRAP activity of up to 5-fold were seen whenC433-conditioned medium was added to isolated avian osteoclasts (FIG.1C). Even greater increases in TRAP activity in response toC433-conditioned medium were seen in giant cells from giant cell tumorsof bone (FIG. 1D). However, these could not be used to monitorpurification, because they were obtained from surgical specimens, andtumor availability was not predictable.

Using a combination of filtration membranes and gel filtrationexperiments, the activity was found to be present in fractions less than5 kDa. FIG. 2 represents a Bio-Gel P2 column showing that the activitywas eluted from this column between the markers for 1000 and 60 daltons.

Further chemical characterization of the activity showed that it wasrelatively heat-stable but completely destroyed by 24 h of ultravioletlight treatment (FIG. 3A). The activity in C433 was not significantlyaffected by treatment with proteases such as aminopeptidase M,carboxypeptidase, or Pronase CB (FIG. 3B). The activity was extractablein ethyl acetate and appeared to behave as an organic molecule. Thus,the activity appeared to be a small nonprotein compound. Sinceprostaglandins and other arachidonic acid metabolites possess thesecharacteristics and are known to have important effects on bone cellfunction, it was determined whether arachidonic acid metabolites couldbe responsible. The initial approach was to determine if production ofthe activity by C433 cells was blocked by inhibitors or arachidonic acidpathway enzymes. These included flurbiprofen and indomethacin whichinhibit prostaglandin synthase, and nordihydroguaiaretic acid, andinhibitor of the lipoxygenases. Flurbiprofen (10⁻⁵ M) and indomethacin(10⁻⁶ M) had no significant effects on production of C433 activity (FIG.3C). The lipoxygenase inhibitor nordihydroguaiaretic acid completelyblocked production of C433 activity in a dose-dependent manner (FIGS. 3Cand 3D).

Since 5-LO inhibition studies implicated 5-LO metabolites as mediatorsof osteoclast activation, a purification protocol was devised forlipoxygenase metabolites of the arachidonic acid pathway. Stability ofthe factors was examined in various solvents useful for HPLCpurification. The activity was stable in ethanol and methanol but not intriethanolamine or pyridine. Acetonitrile was found to be toxic toisolated osteoclasts even after repeated lyophilization, and so methanolwas used for HPLC. After purification using HPLC, bioactivity wasobserved in unretained material and in three fractions corresponding tofraction numbers 9 and 10 (activity 1=25% MeOH), fraction numbers 17 and18 (activity 2=55% MeOH) and fraction number 25 (activity 3=85% MeOH)(FIG. 4A-4C).

The concentrations of 5-LO metabolites in each HPLC fraction weredetermined by stable isotope dilution and gas chromatography-massspectrometry (GC-MS) after hydrogenation and conversion topentafluorobenzyl ester trimethylsilyl ether derivatives. Ionscharacteristic of the product of endogenous 5-LO metabolites (m/z 399)and of the internal standard, [¹³ C₄ ]LTC₄ (m/z 403), were monitoredduring each analysis. Thus, identification of endogenous 5-LO productswas based on the observation of the characteristic ion at theappropriate chromatographic retention time, defined by the internalstandard. The results (FIG. 5) were consistent with the presence of 5-LOmetabolites in each of the HPLC fractions exhibiting osteoclastactivating activity at amounts corresponding (in "LTC₄ equivalents") to35, 83, and 917 pg/ml for the 25, 55, and 85% fractions, respectively.

A commercially available mixture of LTC₄ /LTD₄ was applied to the C₈reverse phase HPLC column under the same conditions as the extractedC433-conditioned medium. LTC_(4/LTD) ₄ eluted in the same fraction asactivity 2 (FIG. 4B). Commercial available 5-HETE was also applied to C₈reverse phase HPLC under the same conditions as the extractedC433-conditioned medium. 5-HETE eluted in the same fraction as activity3 (FIG. 4C). The data are therefore consistent with the identificationof activity 3, the 85% peak, as 5-HETE and activity 2, the 55% peak, asa peptido-leukotriene, either LTC₄, -D₄ or -E₄. Activity 1, the 25%peak, remains unidentified. Commercially available LTB₄ elutes at 80%MeOH, and LTB₄ was not detectable in any fraction by GC/MS analysis.

To confirm that C433-conditioned medium contained these 5-LO metabolitesand that production was hormonally regulated, radioimmunoassays wereperformed for 5-HETE using commercially available kits. C433 cellssecreted large amounts of 5-HETE (Table I) and 1,25-(OH)₂ vitamin D₃increased the production of 5-HETE by C433 cells over control levels.

                  TABLE I                                                         ______________________________________                                        Quantitation of 5-HBTE in C433-conditioned media with and                     without treatment with 1,25-(OH).sub.2 vitamin D.sub.3 (10.sup.-8 M)          A radioinmunoassay (Advanced Magnetics, Inc., Cambridge,                      MA) was performed to measure 5-HETE in C433 conditioned                       media. Units are in pg/ml. Assays were performed in                           duplicate.                                                                                         +1,25-(OH).sub.2 vitamin                                                      D.sub.3                                                  Time      No treatment                                                                             (10.sup.-8 M)                                            ______________________________________                                        0         0.62, 0.51                                                          6         0.91, 0.90  3.36,  3.53                                             12        4.98, 5,71 11.07, 11.68                                             ______________________________________                                    

When activity 2 (putative peptido-leukotriene) and activity 3 (putative5-HETE) were added to the isolated rat osteoclast pit assay inUV-treated C433 conditioned medium, they stimulated bone resorption in amanner similar to that of the untreated C433-conditioned medium (FIGS.6A-6B). Commercially available LTC₄ /LTD₄ and 5-HETE were then tested onboth isolated avian osteoclasts and isolated human giant cells. Withboth avian osteoclasts and human giant cells, LTC₄ /LTD₄ and 5-HETE at10⁻¹⁰ M maximally stimulated TRAP activity (FIGS. 7A and 7B).Commercially available LTC₄ and LTE₄ were tested for capacity tostimulate pit formation by isolated giant cells (FIG. 7C) and isolatedrat osteoclasts (FIG. 7D). Both compounds stimulated isolated resorbingcells at 10⁻¹⁰ M. These commercially available metabolites were alsotested in the murine neonatal calvaria assay and the fetal rat long boneassay, both well recognized assays for measuring the effects ofbone-resorbing cytokines. The metabolites induced resorption in theneonatal murine calvarial assay but not the fetal long bone assay (datanot shown), the same results as that observed with the C433-conditionedmedium (Oreffo et al., 1991).

To determine whether 5-HETE and the peptido-leukotrienes have additiveor synergistic biologic effects, commercially available compounds weretested for TRAP induction in isolated avian osteoclasts. The effects arenot synergistic but appear additive (FIG. 8).

The bone-resorbing activity found in conditioned medium from a stromalcell line isolated from a giant cell tumor of bone (C433) can beascribed to metabolites of the 5-lipoxygenase pathway. These metaboliteswere purified from the conditioned medium of C433 cells, using anosteoclast stimulation assay. Commercially available 5-LO metabolitesmimicked the effects of C433-conditioned medium, LTC₄ /LTD₄, 5-HETE, andC433-conditioned medium caused pit formation by isolated rat osteoclastsand human giant cells and stimulated bone resorption in neonatal mousecalvariae. They also stimulated TRAP activity in avian osteoclasts andhuman giant cells.

Leukotriene (LTB₄) was tested in the mouse calvarial assay and found tostimulate osteoclastic bone resorption even though it was not detectedin the C433 conditioned media. FIG. 8A shows a dose response effect forLTB₄ in the mouse calvarial assay where 10⁻⁸ to 10⁻⁶ M LTB₄ showed astatistically significant degree of resorption stimulation.

Eicosanoids are derived from the oxidative metabolism of arachidonicacid (Smith, 1989; Parker, 1987). These derivatives, which include theprostaglandins, the hydroxyeicosatetraenoic acids, and the leukotrienesare diverse and have powerful but short-lived physiological effects. Ithas been well documented that prostaglandins play a critical role inboth bone resorption and bone formation (Raisz and Martin, 1983), butlittle is known concerning the role of leukotrienes in bone remodeling.Prostaglandins such as prostaglandin E₁ and prostaglandin E₂ in generalare more stable than the leukotrienes and have been shown to inactivateisolated osteoclasts (Chambers and Dunn, 1983).

The C433-conditioned medium maintained activity when stored at 4° C. for3-4 months. However, once purification was initiated, biologicalactivity was quickly lost, especially after HPLC purification. Thestudies suggest the presence of a stabilizing factor in C433-conditionedmedium.

This data indicate that 5-lipoxygenase metabolites stimulate isolatedosteoclasts to resorb bone in vitro and may represent a mechanism bywhich mononuclear cells in human giant cell tumors communicate with thegiant cells. In addition, these results may explain a possible mechanismfor communication between accessory cells and osteoclast activation innormal bone resorption. The analytic evidence supports the presence ofpeptido-leukotriene and 5-HETE, the latter being quantitatively moresignificant in C433-conditioned medium. The unknown eicosanoid ispresent in much smaller amounts and may prove difficult to identify asmuch larger volumes of conditioned medium will be necessary.

EXAMPLE 2 Fetal Rat Long Bone Assay

Fetal rat long bone assays were performed as previously described(Garrett et al, 1990). Briefly, pregnant rats were injected with 200 μCiof ⁴⁵ Ca on the 18th day of gestation. The following day the motherswere euthanized and fetuses removed. The mineralized shafts of the radiiand ulnae are dissected free of cartilaginous tissue and incubated inBGJb media for 24 hours at 37° C. in a humidified atmosphere of 5% CO₂to allow for the exchange of loosely bound ⁴⁵ Ca with stable calcium inthe media. Bones were cultured for a further 48 hours to 120 hours inBGJb media supplemented with 1 mg/ml bovine serum albumin withpenicillin and streptomycin (RIA grade, Sigma, St. Louis, Mo.) in thepresence of test or control substances. Bone resorbing activity wasexpressed as the percentage of the total ⁴⁵ Ca released into the media.

EXAMPLE 3 Isolated Fetal Rat Osteoclasts Assay

Quantitation of the effects of isolated neonatal rat osteoclasts oncalcified matrices was determined using minor modifications of thedisaggregated osteoclast resorption assay as described by Boyde et al(1984). Briefly rat osteoclasts isolated as described by Chambers et al.(1983) were dispersed on prewetted (αMEM plus 5% FBS) slices of spermwhale dentine. Cultures were performed in 48 well-microtiter plates inhumidified air (10% CO₂) at 37° C. in αMEM medium (one slice/well in 200μl medium). Numbers of osteoclasts and resorption lacunae werequantitated as described above for the avian osteoclasts.

EXAMPLE 4 Effects of NDGA on Factor Induced Bone Resorption in theMurine Calvarial Assay

As illustrated in Table II, the murine calvarial assay described inExample 1 was conducted to illustrate the effects of NDGA onfactor-induced bone resorption.

                                      TABLE II                                    __________________________________________________________________________    Effects of NDGA on Factor-Induced Bone Resorption in the Murine               Calvarial                                                                     Assay                                                                                                          Factor +                                                                             %                                     Factor/Conc.)                                                                              Control                                                                             NDGA 10.sup.-5 M                                                                      Factor                                                                              NDGA   Inhibition                            __________________________________________________________________________    IL-1/5 × 10.sup.-10 M                                                                12.1 ± 0.7                                                                       10.6 ± 0.5                                                                         31.2 ± 1.4                                                                        24.6 ± 1.6*                                                                      32.8                                               13.4 ± 0.4                                                                       14.4 ± 0.9                                                                         27.7 ± 1.2                                                                        23.4 ± 1.4*                                                                      30.0                                  TNP/2 × 10.sup.-10 M                                                                 16.9 ± 1.3                                                                       14.2 ± 1.0                                                                         36.1 ± 2.3                                                                        27.8 ± 1.7*                                                                      43.2                                               16.4 ± 0.8                                                                       16.0 ± 1.8                                                                         39.7 ± 1.9                                                                        31.9 ± 1.5*                                                                      33.5                                  Lymphotoxin/5 × 10.sup.-9 M                                                          18.2 ± 0.7                                                                       15.7 ± 0.5                                                                         35.0 ± 1.5                                                                        26.8 ± 1.3*                                                                      48.8                                               19.8 ± 2.6                                                                       17.4 + 1.3                                                                            42.8 ± 1.2                                                                        35.5 ± 1.8*                                                                      31.7                                  PTH/4 ng/ml  16.0 ± 0.9                                                                       15.0 ± 0.9                                                                         37.0 ± 1.4                                                                       30.6 ± 1.8                                                                        30.5                                               14.7 ± 0.4                                                                       13.0 ± 1.1                                                                         39.9 ± 2.3                                                                        31.2 ± 0.9*                                                                      34.5                                               15.7 ± 0.6                                                                       12.8 ± 1.3                                                                         29.8 ± 2.9                                                                       28.6 ± 2.7                                                                         8.5                                  1,25D.sub.3 /10.sup.-10 M                                                                  16.9 ± 1.3                                                                       14.2 ± 1.0                                                                         35.3 ± 1.5                                                                       31.7 ± 2.7                                                                        19.6                                               18.7 ± 1.3 31.8 ± 2.2                                                                       29.2 ± 3.9                                                                        19.8                                               16.0 ± 0.9                                                                       15.0 ± 0.9                                                                         37.0 ± 1.4                                                                       32.5 ± 1.4                                                                        24.1                                  __________________________________________________________________________     *=significantly different;                                                    p < 0.05. (As also in later tables.)                                     

These data show that NDGA significantly inhibits bone resorption due tothe factors: interleukin-1 (IL-1), tumor necrosis factor (TNF) andlymphotoxin. One out of three experiments showed significant inhibitionof parathyroid hormone (PTH)-induced resorption but no significance wasobserved for 1,25(OH)₂ vitamin D₃ -induced resorption.

EXAMPLE 5 Effects of NDGA on LPS-Induced Bone Resorption in the MurineCalvarial Assay

Bacterial lipopolysaccharide (LPS) is known to induce bone resorptionand may be particularly relevant in certain inflammatory conditions. Theeffects of NDGA on lipopolysaccharide-induced bone resorption weremeasured in the murine calvarial assay described in Example 1. Greaterthan 50% NDGA inhibition of bone resorption was noted for two differentpreparations of lipopolysaccharide. A summation of these results is seenin Table III.

                                      TABLE III                                   __________________________________________________________________________    Effects of NDGA on LPS Induced Bone Resorption in Murine Calvarial Assay      LPS                               %                                           Used  Control                                                                             NDGA 10.sup.-5M                                                                       LPS   LPS + NDGA                                                                            Inhibition                                  __________________________________________________________________________    AaY4 LPS                                                                            15.4 ± 0.53                                                                      12.7 ± 0.8                                                                         31.3 ± 1.4                                                                       22.2 ± 2.8*                                                                        57.2                                        50 mg/ml                                                                      WR14 LPS                                                                            12.8 ± 0.8                                                                       10.9 ± 0.5                                                                         35.2 ± 1.3                                                                       22.3 ± 2.3*                                                                        57.6                                        50 ng/ml                                                                      __________________________________________________________________________

Significant inhibition of the resorption effects of both bacteriallipopolysaccharide (LPS) preparations was observed with NDGA.

EXAMPLE 6 Effects of 5LO Inhibitor MK886 on Factor-induced BoneResorption in the Murine Calvarial Assay

The effects of 5LO inhibitor MK886 on bone resorption induced by 1,25dihydroxyvitamin D₃ and bone resorption induced by Interleukin-1 weremeasured in the murine calvarial assay (as described in Example 1). Theresults of these measurements are shown in Table IV.

                                      TABLE IV                                    __________________________________________________________________________    Effects of MK886 on Factor Induced Bone Resorption                            in Murine Calvarial Assay                                                          MK886 2.5     IL-1                                                       Control                                                                            μg/ml                                                                            1,25D.sub.3 10.sup.-10 M                                                              1 × 10.sup.-10 M                                                                      Inhibition                                   __________________________________________________________________________    +                         15.4 ± 0.6                                            +                    9.6 ± 0.3                                                   +              37.2 ± 0.7                                            +     +               32.1 ± 2.4*                                                                      23.4                                         +                         21.5 ± 1.6                                            +                    14.2 ± 0.7                                                          +      38.9 ± 2.4                                            +             +      34.9 ± 1.7                                                                        23.0                                         __________________________________________________________________________

As shown in Table IV, the 5LO inhibitor MK886 significantly inhibited1,25 dihydroxyvitamin D₃ -induced bone resorption. However, asignificant inhibition of Interleukin-1 induced bone resorption was notnoted.

EXAMPLE 7 Effects of NDGA and MK886 on Lipopolysaccharide-stimulatedResorption in the Murine Calvarial Assay

The effects of NDGA and 5LO inhibitor MK886 were measured onlipopolysaccharide-stimulated bone resorption in the murine calvarialassay described in Example 1. Table V summarizes the results obtainedwith this study of bone resorption.

                                      TABLE V                                     __________________________________________________________________________    Effects of NDGA and MK886 in Combination on LPS Stimulated                    Resorption in the Murine Calvarial Assay                                                    AAY4 LPS                                                                            WR14 LPS                                                                            Ca.sup.45                                                                            %                                            C MK886                                                                             NDGA 10.sup.-5 M                                                                      25 μg/ml                                                                         50 ng/ml                                                                            Release                                                                              Inhibition                                   __________________________________________________________________________    +                         15.4 ± 1.4                                                     +           24.5 ± 0.7                                         +           +           22.6 ± 1.5                                                                        20.9                                               +       +            13.6 ± 0.4*                                                                      119.8                                                            +     33.8 ± 1.1                                         +                 +     32.8 ± 1.8                                                                        8.2                                                +             +      26.3 ± 0.4*                                                                      40.8                                         __________________________________________________________________________

As shown in Table V, MK886 only mildly inhibited bone resorption inducedby AaY4 LPS or WR14 LPS. With NDGA, LPS-induced resorption wassignificantly inhibited.

EXAMPLE 8 Effects of ZM 230,487 on PTH Induced Bone Resorption in theMurine Calvarial Assay (% Total Calcium Released)

The murine calvarial assay described in Example 1 was utilized to testthe effects of the 5LO inhibitor ZM230487 on PTH-induced boneresorption. The results of this assay are seen in Table VI.

                  TABLE VI                                                        ______________________________________                                        Effects of ZM 230,487 on PTH Induced Bone Resorption                          in the Murine Calvarial Assay                                                                         % Inhibition                                          ______________________________________                                        Control          12.1 ± 0.8                                                ZM 230,487 (100 ng/ml)                                                                         11.1 ± 0.6                                                PTH (10 ng/ml)   30.5 ± 2.4                                                PTH + 100 ng/ml ZM                                                                              17.7 ± 1.7*                                                                            69.8                                            230,487                                                                       PTH + 50 ng/ml ZM 230,487                                                                       23.3 ± 3.3*                                                                            40.1                                            ______________________________________                                    

As shown in Table VI, a significant inhibition of bone resorptionoccurred at both concentrations of this 5LO inhibitor.

EXAMPLE 9 Effects of ZM 230,487 on Factor-Induced Bone Resorption MurineCalvarial Assay

In the murine calvarial system of Example 1, the effects of the 5LOinhibitor ZM230487 was studied on bone resorption induced byInterleukin-1, tumor necrosis factor and lipopolysaccharide. The resultsare shown in Table VII.

                                      TABLE VII                                   __________________________________________________________________________    Effects of ZM 230,487 on Factor Induced Bone Resorption                       Murine Calvarial Assay                                                        Factor       ZM 230,487  Factor +                                                                             %                                             (Conc.)                                                                              Control                                                                             100 ng/ml                                                                           Factor                                                                              ZM 230,487                                                                           Inhibition                                    __________________________________________________________________________    IL-10  15.2 ± 1.0                                                                       13.2 ± 0.4                                                                       32.0 ± 0.8                                                                        18.3 ± 0.4*                                                                      81.7                                          5 × 10.sup.-10 M                                                        TNF                27.7 ± 1.8                                                                        17.4 ± 0.5*                                                                      82.4                                          5 × 10.sup.-10 M                                                        LPS WR14                                                                             13.5 ± 0.4                                                                       12.6 ± 0.3                                                                       39.5 ± 1.1                                                                       18.5 ± 0.5                                                                        69.8                                          50 ng/ml                                                                      __________________________________________________________________________

ZM 230,847, at 100 ng/ml; 25-fold less than MK886, 50-fold less thanNDGA, as noted in other Examples) significantly inhibits bone resorptiondue to IL-1, TNF and LPS WR 14.

EXAMPLE 10 The Effects of NDGA, MK886 and ZM230487 on PTH andPTHrP-Induced Bone Resorption

The effects of NDGA, MK886 and ZM230487 on PTH and PTHrP-induced boneresorption in the isolated fetal rat osteoclast assay described inExample 3 were measured. The results of these experiments are seen inFIGS. 12A-12D. It should be noted that inhibition of the bone resorptionwas seen with all three 5LO inhibitors, the order of inhibitoryeffectiveness, as noted in FIGS. 12A-12D, was ZM230487>MK886>NDGA.

EXAMPLE 11 Effect of the 5LO Inhibitor ZM230,487 on Rat Osteoclast PitFormation

The rat osteoclast Assay of Example 3 was also used in the context oflipopolysaccharide-induced or 1,25-dihydroxyvitamin D₃ -induced boneresorption. The results of these assays are seen in FIGS. 13A and 13B.It should be noted that the 5LO inhibitor ZM230487 effectively inhibitedthe induction of bone resorption by lipopolysaccharide, tumor necrosisfactor, and 1,25 dihydroxyvitamin D₃.

The following citations are incorporated in pertinent fact by referenceherein for the reasons cited in the above text.

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What is claimed is:
 1. A method for inhibiting bone resorptioncomprising administering1-ethyl-6-[fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl)phenoxy]methyl-quinol-2-oneto a subject in an amount suppressing production of anosteoclast-stimulating factor.
 2. The method of claim 1 where the factoris a leukotriene, peptidoleukotriene, or 5-hydroxyeicosatetraenoic acid.3. The method of claim 1 where the bone resorption is related toperiodontal disease, osteoporosis, estrogen deficiency, Paget's disease,inflammatory bone loss, bone malignancy or hyperparathyroidism.
 4. Themethod of claim 1 where the administering is enteral, parenteral ortopical.
 5. The method of claim 1 where the amount is from 0.1 to 10mg/kg body weight/day.
 6. A method for inhibiting bone resorption in apatient with periodontal disease, osteoporosis, estrogen deficiency,Paget's disease, inflammatory bone loss, bone malignancy orhyperparathyroidism, the method comprising administering1-ethyl-6-[fluoro-5-(4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl)phenoxy]methyl-quinol-2-onein an amount suppressing production of an osteoclast-stimulating factor.7. The method of claim 6 where the factor is a leukotriene,peptidoleukotriene, or 5-hydroxyeicosatetraenoic acid.
 8. The method ofclaim 6 where the administering is enteral, parenteral or topical. 9.The method of claim 6 where the amount is from 0.1 to 10 mg/kg bodyweight/day.
 10. The method of claim 6 where a patient with periodontaldisease is being treated and the administration is topical.
 11. Themethod of claim 6 where a patient with osteoporosis is being treated andthe administration is enteral.